The techniques of plant biotechnology have improved during the last ten years so that most of the crop species important to the mankind can be transformed today. This has led to a situation where a continuously increasing number of crop species have been transformed with a continuously increasing number of traits. This together with the concerns of environmental impacts of genetically modified (GM) crops has created a clear need for a new generation of GM-crops having a reduced probability of transgene flow among crops and their relatives. Accordingly, several research groups around the world are currently engaged in developing techniques for gene containment in transgenic crops. Henry Daniel 2002 (Molecular Strategies for Gene Containment in Transgenic Crops. Nature Biotech 20: 591–585) gives a good review of the techniques available today.
The molecular approaches for control of transgene flow can basically be divided into to two groups: one-factor systems and two-factor systems. The one-factor techniques use a negative selection factor to prevent any plant carrying the transgene to interbreed with wild type relatives or with their own offspring. Examples of one-factor technologies are male sterility described in U.S. patent application No. US2002157129, chloroplast transformation described by Scott and Wilkinson in Nat. Biotechnol. 17. 390–392 (1999), technology launched by Monsanto and known as ‘terminator technology’ described in U.S. Pat. No. 5,723,765 and tandem mitigation concept described by Gressel in Trend in Biotech. 17: 361–366.
The main limitation of one-factor techniques is that they may not offer absolute transgene containment. The negative selection eliminates transgene from population of plants in course of several generations. Acting as a negative selection factor, male sterility and chloroplast transformation decrease the reproduction capacity of transgenic plants by limiting pollen spread. These methods however, do not prevent transgene flow through seed shatter. The ‘terminator’ technology prevents gene flow only when killer (or terminator) gene is activated. After the activation of killer gene (negative selection factor) seeds of the next progeny are incapable to germinate and, therefore the plants cannot be propagated further. If the killer gene is not activated the transgene insert can freely flow because the killer gene remains silent.
The idea of tandem mitigation technology is to use genes, which are adverse for wild plants but neutral for cultured transgenic relatives. Because there are no genes absolutely perfect for such purposes, the technology can only exploit genes that reduce the vitality/reproductivity of wild relatives carrying the transgene insert. Therefore the limitation of this technology is that several generations are needed to remove the transgene from population.
Recently, two-factor concepts of molecular control have been proposed to significantly reduce a probability of transgene introgression into a population of sexually compatible plants.
Basically two-factor technologies use the negative selection factor (BC or EC), which absolutely prevents the transgene flow. This is made possible by using another rescuing factor, which represses the action of the first factor, disrupt killer gene or recovers the blocked function of the plant.
International patent publication WO9403619 (Bright et al.) describes a method, where disrupter gene (negative selection factor—BC) disrupts the transgene of interest or its promoter (by Cre recombinase) or kills the plant. Chemically inducible repressor gene represses the promoter of disrupter gene.
International patent publication WO0037660 (Fabijanski et al.) describes the system where lethal gene (BC) is linked to a transgene of interest. Repressor gene (RC) is placed into another allelic (sister) or non-allelic chromosome. Second pair of lethal and repressor genes can be placed in opposite order in the same inserts.
International patent publication WO02064801 (Kuvhshinov et al.) describes a system, where Excision construct EC is linked to the TGI. The EC excises the whole insert from the genome of the host organism under natural conditions. Artificially activated repression construct RC represses the action of the EC and saves the transgenic insert in the host genome. This system removes the entire transgene insert and leaves the host genome free from the foreign genes. Thus, transgenic plants can produce non-transgenic seeds.
Although the described prior art gives advanced alternatives to control transgene flow, none of the prior art resolves the problem of BC being inactivated by mutagenesis. This can happen approximately with a frequency of 10−6. In practice this means once in each middle size field plot during a growth season. The present invention markedly decreases the probability of BC to become inactivated.
U.S. patent application Ser. No. 09/617,543 (Kuvshinov et al.) discloses a two-factor system called RBF (recoverable block of function) system. RBF system comprises a blocking construct (BC) linked to a transgene of interest (TGI) and a recovering construct (RC). According to this invention BC blocks a vital physiological or molecular function of the host plant through developmental or organ specific expression. The RC is induced by an externally controllable stimulus and when induced it recovers the function previously blocked by the BC. Even if this system is a huge step ahead in transgene containment techniques it as such does not remove the possibility of reorganization of genomic DNA in the segregating progenies.
This problem is resolved with the invention according to the present disclosure. The present invention minimizes reorganization of genomic DNA in a RBF system similar to that described in U.S. patent application Ser. No. 09/617,543 now U.S. Pat. No. 6,849,776.
According to the present disclosure the BC is placed into an intron of the TGI, thereby providing an inseparable genetic linkage between the BC and the TGI. This arrangement minimizes the probability of crossing-overs between the BC and the TGI and thereby prevents them being segregated.
Furthermore, this arrangement minimizes probability of large mutations of the BC without destroying the TGI simultaneously. Therefore, this disclosure resolves another problem the prior art includes; i.e. mutated BC would probably not block the reproduction of the transgenic plant and therefore the containment of the transgene would be incomplete if the TGI was not destroyed too.